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Carotenoid pathway engineering in carrot and functional characterization of cytochrome P450 carotenoid hydroxylases Kim, Ji-Eun


Carotenoid pigments are synthesized by bacteria, fungi and plants for photosynthesis and photoprotection. The ketocarotenoid astaxanthin is a strong anti-oxidant and has also been exploited as fish feed additive to provide the attractive red colour in salmon flesh. Astaxanthin cannot be produced efficiently from natural sources and has to be chemically synthesized. I explored the feasibility of synthesizing ketocarotenoids in transgenic carrots. Using light and transmission electron microscopy, I confirmed that the biosynthesis and accumulation of carotenoids mostly occurred in the chromoplasts in the phloem tissue of carrot roots, and also found that increased accumulation of β-carotene in roots of the High Carotene Mass (HCM) carrot variety was achieved by increased β-carotene content in the chromoplasts and not by increasing the number of chromoplasts. An Agrobacterium binary vector system with the CaMV35S promoter directing expression of a crtO ketolase gene (isolated from the algal Haematococcus pluvialis) was used to transform HCM carrots. PCR analysis of genomic DNA from regenerated plants confirmed the insertion of the crtO gene in the carrot genome. Further work by our research team has detected novel ketocarotenoids: Astaxanthin, adonixanthin, adonirubin, canthaxanthin, β-cryptoxanthin, and echinenone in the transformed carrots. It is of interest to examine the working relationship between the ketolase and hydroxylases in carotenoid biosynthetic pathways. I started by generating a single gene crtO transgenic line in Arabidopsis, three single gene carotenoid hydroxylase overexpression lines (AtB1, CYP97A3, and CYP97C1), and a single gene candidate carotenoid hydroxylase overexpression line (CYP97B3). HPLC-aided profiling of the carotenoid products in the leaves of crtO ketolase transgenic plants indicated no ketocarotenoid production. The three hydroxylase overexpression lines confirmed and also clarified the roles of the three hydroxylases in the carotenoid synthesis pathways in Arabidopsis . While I found that CYP97B3 has β-ring hydroxylase activity and allowed biosynthesis of zeaxanthin and β-cryptoxanthin in E.coli , the CYP97B3 overexpression line provided evidence for the first time that CYP97B3 gene product has hydroxylase activity primarily in the α-carotene pathway. I also co-transformed the crtO gene with each of the four hydroxylase genes to generate four co-transformants. In all co-transformants, hydroxylase activities in the α-carotene pathway were enhanced at the cost of hydroxylation activities in the β-carotene pathway.

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